Novel Pseudomonas Species Prevent the Growth of the Phytopathogenic Fungus Aspergillus flavus.

In response to the escalating demand for sustainable agricultural methodologies, the utilization of microbial volatile organic compounds (VOCs) as antagonists against phytopathogens has emerged as a viable eco-friendly alternative. Microbial volatiles exhibit rapid diffusion rates, facilitating prompt chemical interactions. Moreover, microorganisms possess the capacity to emit volatiles constitutively, as well as in response to biological interactions and environmental stimuli. In addition to volatile compounds, these bacteria demonstrate the ability to produce soluble metabolites with antifungal properties, such as APE Vf, pyoverdin, and fragin. In this study, we identified two Pseudomonas strains (BJa3 and MCal1) capable of inhibiting the in vitro mycelial growth of the phytopathogenic fungus Aspergillus flavus, which serves as the causal agent of diseases in sugarcane and maize. Utilizing GC/MS analysis, we detected 47 distinct VOCs which were produced by these bacterial strains. Notably, certain volatile compounds, including 1-heptoxydecane and tridecan-2-one, emerged as primary candidates for inhibiting fungal growth. These compounds belong to essential chemical classes previously documented for their antifungal activity, while others represent novel molecules. Furthermore, examination via confocal microscopy unveiled significant morphological alterations, particularly in the cell wall, of mycelia exposed to VOCs emitted by both Pseudomonas species. These findings underscore the potential of the identified BJa3 and MCal1 Pseudomonas strains as promising agents for fungal biocontrol in agricultural crops.

Improving annotation propagation on molecular networks through random walks: introducing ChemWalker.

MOTIVATION: Annotation of the mass signals is still the biggest bottleneck for the untargeted mass spectrometry analysis of complex mixtures. Molecular networks are being increasingly adopted by the mass spectrometry community as a tool to annotate large-scale experiments. We have previously shown that the process of propagating annotations from spectral library matches on molecular networks can be automated using Network Annotation Propagation (NAP). One of the limitations of NAP is that the information for the spectral matches is only propagated locally, to the first neighbor of a spectral match. Here, we show that annotation propagation can be expanded to nodes not directly connected to spectral matches using random walks on graphs, introducing the ChemWalker python library. RESULTS: Similarly to NAP, ChemWalker relies on combinatorial in silico fragmentation results, performed by MetFrag, searching biologically relevant databases. Departing from the combination of a spectral network and the structural similarity among candidate structures, we have used MetFusion Scoring function to create a weight function, producing a weighted graph. This graph was subsequently used by the random walk to calculate the probability of 'walking' through a set of candidates, departing from seed nodes (represented by spectral library matches). This approach allowed the information propagation to nodes not directly connected to the spectral library match. Compared with NAP, ChemWalker has a series of improvements, on running time, scalability and maintainability and is available as a standalone python package. AVAILABILITY AND IMPLEMENTATION: ChemWalker is freely available at https://github.com/computational-chemical-biology/ChemWalker. CONTACT: ridasilva@usp.br. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Metabolomics Analysis of Bacterial Pathogen Burkholderia thailandensis and Mammalian Host Cells in Co-culture.

The Tier 1 HHS/USDA Select Agent Burkholderia pseudomallei is a bacterial pathogen that is highly virulent when introduced into the respiratory tract and intrinsically resistant to many antibiotics. Transcriptomic- and proteomic-based methodologies have been used to investigate mechanisms of virulence employed by B. pseudomallei and Burkholderia thailandensis, a convenient surrogate; however, analysis of the pathogen and host metabolomes during infection is lacking. Changes in the metabolites produced can be a result of altered gene expression and/or post-transcriptional processes. Thus, metabolomics complements transcriptomics and proteomics by providing a chemical readout of a biological phenotype, which serves as a snapshot of an organism's physiological state. However, the poor signal from bacterial metabolites in the context of infection poses a challenge in their detection and robust annotation. In this study, we coupled mammalian cell culture-based metabolomics with feature-based molecular networking of mono- and co-cultures to annotate the pathogen's secondary metabolome during infection of mammalian cells. These methods enabled us to identify several key secondary metabolites produced by B. thailandensis during infection of airway epithelial and macrophage cell lines. Additionally, the use of in silico approaches provided insights into shifts in host biochemical pathways relevant to defense against infection. Using chemical class enrichment analysis, for example, we identified changes in a number of host-derived compounds including immune lipids such as prostaglandins, which were detected exclusively upon pathogen challenge. Taken together, our findings indicate that co-culture of B. thailandensis with mammalian cells alters the metabolome of both pathogen and host and provides a new dimension of information for in-depth analysis of the host-pathogen interactions underlying Burkholderia infection.

Microbial and Nonvolatile Chemical Diversities of Chinese Dark Teas Are Differed by Latitude and Pile Fermentation.

Understanding the microbial and chemical diversities, as well as what affects these diversities, is important for modern manufacturing of traditional fermented foods. In this work, Chinese dark teas (CDTs) that are traditional microbial fermented beverages with relatively high sample diversity were collected. Microbial DNA amplicon sequencing and mass spectrometry-based untargeted metabolomics show that the CDT microbial ß diversity, as well as the nonvolatile chemical α and ß diversities, is determined by the primary impact factors of geography and manufacturing procedures, in particular, latitude and pile fermentation after blending. A large number of metabolites sharing between CDTs and fungi were discovered by Feature-based Molecular Networking (FBMN) on the Global Natural Products Social Molecular Networking (GNPS) web platform. These molecules, such as prenylated cyclic dipeptides and B-vitamins, are functionally important for nutrition, biofunctions, and flavor. Molecular networking has revealed patterns in metabolite profiles on a chemical family level in addition to individual structures.

Metabolic Profiling of Interspecies Interactions During Sessile Bacterial Cultivation Reveals Growth and Sporulation Induction in Paenibacillus amylolyticus in Response to Xanthomonas retroflexus.

The toolbox available for microbiologists to study interspecies interactions is rapidly growing, and with continuously more advanced instruments, we are able to expand our knowledge on establishment and function of microbial communities. However, unravelling molecular interspecies interactions in complex biological systems remains a challenge, and interactions are therefore often studied in simplified communities. Here we perform an in-depth characterization of an observed interspecies interaction between two co-isolated bacteria, Xanthomonas retroflexus and Paenibacillus amylolyticus. Using microsensor measurements for mapping the chemical environment, we show how X. retroflexus promoted an alkalization of its local environment through degradation of amino acids and release of ammonia. When the two species were grown in proximity, the modified local environment induced a morphological change and growth of P. amylolyticus followed by sporulation. 2D spatial metabolomics enabled visualization and mapping of the degradation of oligopeptide structures by X. retroflexus and morphological changes of P. amylolyticus through e.g. the release of membrane-associated metabolites. Proteome analysis and microscopy were used to validate the shift from vegetative growth towards sporulation. In summary, we demonstrate how environmental profiling by combined application of microsensor, microscopy, metabolomics and proteomics approaches can reveal growth and sporulation promoting effects resulting from interspecies interactions.

Feature-based molecular networking in the GNPS analysis environment.

Molecular networking has become a key method to visualize and annotate the chemical space in non-targeted mass spectrometry data. We present feature-based molecular networking (FBMN) as an analysis method in the Global Natural Products Social Molecular Networking (GNPS) infrastructure that builds on chromatographic feature detection and alignment tools. FBMN enables quantitative analysis and resolution of isomers, including from ion mobility spectrometry.

Differences in Cystic Fibrosis-Associated Burkholderia spp. Bacteria Metabolomes after Exposure to the Antibiotic Trimethoprim.

The Burkholderia cepacia complex is a group of closely related bacterial species with large genomes that infect immunocompromised individuals and those living with cystic fibrosis. Some of these species are found more frequently and cause more severe disease than others, yet metabolomic differences between these have not been described. Furthermore, our understanding of how these species respond to antibiotics is limited. We investigated the metabolomics differences between three most prevalent Burkholderia spp. associated with cystic fibrosis: B. cenocepacia, B. multivorans, and B. dolosa in the presence and absence of the antibiotic trimethoprim. Using a combination of supervised and unsupervised metabolomics data visualization and analysis tools, we describe the overall differences between strains of the same species and between species. Specifically, we report, for the first time, the role of the pyomelanin pathway in the metabolism of trimethoprim. We also report differences in the detection of known secondary metabolites such as fragin, ornibactin, and N-acylhomoserine lactones and their analogs in closely related strains. Furthermore, we highlight the potential for the discovery of new secondary metabolites in clinical strains of Burkholderia spp. The metabolomics differences described in this study highlight the personalized nature of closely related Burkholderia strains.

Assessing specialized metabolite diversity of Alnus species by a digitized LC-MS/MS data analysis workflow.

Alnus spp. (Betulaceae) have been used for treatments of hemorrhage, burn injuries, antipyretic fever, diarrhea, and alcoholism in traditional medicines. In this study, a digitized LC-MS/MS data analysis workflow was applied to provide an overview on chemical diversity of 15 Alnus extracts prepared from bark, twigs, leaves, and fruits of A. japonica, A. firma, A. hirsuta, and A. hirsuta var. sibirica. Most of the MS/MS spectra could be putatively annotated based on library matching, in silico fragmentation, and substructural topic modeling. The putative annotation allowed us to discriminate the extracts into three chemotypes based on dominant chemical scaffolds: diarylheptanoids, flavonoids or tannins. This high-throughput chemical annotation was correlated with α-glucosidase inhibition data of extracts, and it allowed us to identify gallic acid as the major active compound of A. firma.

Untargeted mass spectrometry-based metabolomics approach unveils molecular changes in raw and processed foods and beverages.

In our daily lives, we consume foods that have been transported, stored, prepared, cooked, or otherwise processed by ourselves or others. Food storage and preparation have drastic effects on the chemical composition of foods. Untargeted mass spectrometry analysis of food samples has the potential to increase our chemical understanding of these processes by detecting a broad spectrum of chemicals. We performed a time-based analysis of the chemical changes in foods during common preparations, such as fermentation, brewing, and ripening, using untargeted mass spectrometry and molecular networking. The data analysis workflow presented implements an approach to study changes in food chemistry that can reveal global alterations in chemical profiles, identify changes in abundance, as well as identify specific chemicals and their transformation products. The data generated in this study are publicly available, enabling the replication and re-analysis of these data in isolation, and serve as a baseline dataset for future investigations.

Molecular and Microbial Microenvironments in Chronically Diseased Lungs Associated with Cystic Fibrosis.

To visualize the personalized distributions of pathogens and chemical environments, including microbial metabolites, pharmaceuticals, and their metabolic products, within and between human lungs afflicted with cystic fibrosis (CF), we generated three-dimensional (3D) microbiome and metabolome maps of six explanted lungs from three cystic fibrosis patients. These 3D spatial maps revealed that the chemical environments differ between patients and within the lungs of each patient. Although the microbial ecosystems of the patients were defined by the dominant pathogen, their chemical diversity was not. Additionally, the chemical diversity between locales in the lungs of the same individual sometimes exceeded interindividual variation. Thus, the chemistry and microbiome of the explanted lungs appear to be not only personalized but also regiospecific. Previously undescribed analogs of microbial quinolones and antibiotic metabolites were also detected. Furthermore, mapping the chemical and microbial distributions allowed visualization of microbial community interactions, such as increased production of quorum sensing quinolones in locations where Pseudomonas was in contact with Staphylococcus and Granulicatella, consistent with in vitro observations of bacteria isolated from these patients. Visualization of microbe-metabolite associations within a host organ in early-stage CF disease in animal models will help elucidate the complex interplay between the presence of a given microbial structure, antibiotics, metabolism of antibiotics, microbial virulence factors, and host responses.IMPORTANCE Microbial infections are now recognized to be polymicrobial and personalized in nature. Comprehensive analysis and understanding of the factors underlying the polymicrobial and personalized nature of infections remain limited, especially in the context of the host. By visualizing microbiomes and metabolomes of diseased human lungs, we reveal how different the chemical environments are between hosts that are dominated by the same pathogen and how community interactions shape the chemical environment or vice versa. We highlight that three-dimensional organ mapping methods represent hypothesis-building tools that allow us to design mechanistic studies aimed at addressing microbial responses to other microbes, the host, and pharmaceutical drugs.

Investigation of Premyrsinane and Myrsinane Esters in Euphorbia cupanii and Euphobia pithyusa with MS2LDA and Combinatorial Molecular Network Annotation Propagation.

The species Euphorbia pithyusa and Euphorbia cupanii are two closely related Mediterranean spurges for which their taxonomic relationships are still being debated. Herein, the diterpene ester content of E. cupanii was investigated using liquid chromatography coupled to tandem mass spectrometry. The use of molecular networking coupled to unsupervised substructure annotation ( MS2LDA) indicated the presence of new premyrsinane/myrsinane diterpene esters in the E. cupanii fractions. A structure-guided isolation procedure yielded 16 myrsinane (11a-h, 12, and 13) and premyrsinane esters (14a-c and 15a-c), along with four 4ß-phorbol esters (16a-c and 17) that showed inhibitory activity against chikungunya virus replication. The structures of the 16 new compounds (11a-c, 11h, 12, 13, 14a-c, 15a-c, 16a-c, and 17) were characterized by NMR spectroscopy and X-ray crystallography. To further uncover the diterpene ester content of these two species, the concept of combinatorial network annotation propagation (C-NAP) was developed. By leveraging the fact that the diterpene esters of Euphorbia species are made up of limited building blocks, a combinatorial database of theoretical structures was created and used for C-NAP that made possible the annotation of 123 premyrsinane or myrsinane esters, from which 74% are not found in any compound database.

In silico annotation of discriminative markers of three Zanthoxylum species using molecular network derived annotation propagation.

In liquid chromatography-mass spectrometry (LC-MS) metabolomics, data matrices with up to thousands of variables for each ion peak are subjected to multivariate analysis (MVA) to assess the homogeneity between samples. The large dimensions of LC/MS datasets hinder the identification of the discriminant or the metabolic markers. In the present study, the molecular network (MN) approach and two in silico annotation tools, network annotation propagation (NAP) and the hierarchical chemical classification method, ClassyFire, were used to annotate the metabolites of three Zanthoxylum species, Z. bungeanum, Z. schinifolium and Z. piperitum. The in silico annotation results of the MN nodes and the MVA variables were combined and visualized in loading plots. This approach helped intuitive detection of the variables that greatly contributed to the separation of the samples in the score plot as discriminant or metabolic markers, thereby allowing rapid annotation of two flavanone derivatives.

Initial Development toward Non-Invasive Drug Monitoring via Untargeted Mass Spectrometric Analysis of Human Skin.

Drug monitoring is crucial for providing accurate and effective care; however, current methods (e.g., blood draws) are inconvenient and unpleasant. We aim to develop a non-invasive method for the detection and monitoring of drugs via human skin. The initial development toward this aim required information about which drugs, taken orally, can be detected via the skin. Untargeted liquid chromatography-mass spectrometry (LC-MS) was used as it was unclear if drugs, known drug metabolites, or other transformation products were detectable. In accomplishing our aim, we analyzed samples obtained by swabbing the skin of 15 kidney transplant recipients in five locations (forehead, nasolabial area, axillary, backhand, and palm), bilaterally, on two different clinical visits. Untargeted LC-MS data were processed using molecular networking via the Global Natural Products Social Molecular Networking platform. Herein, we report the qualitative detection and location of drugs and drug metabolites. For example, escitalopram/citalopram and diphenhydramine, taken orally, were detected in forehead, nasolabial, and hand samples, whereas N-acetyl-sulfamethoxazole, a drug metabolite, was detected in axillary samples. In addition, chemicals associated with environmental exposure were also detected from the skin, which provides insight into the multifaceted chemical influences on our health. The proof-of-concept results presented support the finding that the LC-MS and data analysis methodology is currently capable of the qualitative assessment of the presence of drugs directly via human skin.

Comprehensive mass spectrometry-guided phenotyping of plant specialized metabolites reveals metabolic diversity in the cosmopolitan plant family Rhamnaceae.

Plants produce a myriad of specialized metabolites to overcome their sessile habit and combat biotic as well as abiotic stresses. Evolution has shaped the diversity of specialized metabolites, which then drives many other aspects of plant biodiversity. However, until recently, large-scale studies investigating the diversity of specialized metabolites in an evolutionary context have been limited by the impossibility of identifying chemical structures of hundreds to thousands of compounds in a time-feasible manner. Here we introduce a workflow for large-scale, semi-automated annotation of specialized metabolites and apply it to over 1000 metabolites of the cosmopolitan plant family Rhamnaceae. We enhance the putative annotation coverage dramatically, from 2.5% based on spectral library matches alone to 42.6% of total MS/MS molecular features, extending annotations from well-known plant compound classes into dark plant metabolomics. To gain insights into substructural diversity within this plant family, we also extract patterns of co-occurring fragments and neutral losses, so-called Mass2Motifs, from the dataset; for example, only the Ziziphoid clade developed the triterpenoid biosynthetic pathway, whereas the Rhamnoid clade predominantly developed diversity in flavonoid glycosides, including 7-O-methyltransferase activity. Our workflow provides the foundations for the automated, high-throughput chemical identification of massive metabolite spaces, and we expect it to revolutionize our understanding of plant chemoevolutionary mechanisms.

Computational Removal of Undesired Mass Spectral Features Possessing Repeat Units via a Kendrick Mass Filter.

Polymers are a common component of chemical background which complicates data analysis and can impair interpretation. Undesired chemical background cannot always be addressed via pre-analytical methods, chromatography, or existing data processing methods. The Kendrick mass filter (KMF) is presented for the computational removal of undesired signals present in MS1 spectra. The KMF is analogous to mass defect filtering but utilizes homology information via Kendrick mass scaling in combination with chromatographic retention time and the number of observed signals. The KMF is intended to assist in situations in which current data processing methods to remove background, e.g., blank subtraction, are either not possible or effective. The major parameters affecting KMF were investigated using PEG 400 and NIST standard reference material 1950 (metabolites in human plasma). Further exploration of the KMF performance was tested using an extract of a swab known to contain polymers. An illustrative real-world example of skin analysis with polymeric signal is discussed. The KMF is also able to provide a high-level view of the compositionality of data regarding the presence of signals with repeat units and indicate the presence of different polymers. Graphical Abstract ᅟ.

Targeted Isolation of Neuroprotective Dicoumaroyl Neolignans and Lignans from Sageretia theezans Using in Silico Molecular Network Annotation Propagation-Based Dereplication.

The integration of LC-MS/MS molecular networking and in silico MS/MS fragmentation is an emerging method for dereplication of natural products. In the present study, a targeted isolation of natural products using a new in silico-based annotation tool named Network Annotation Propagation (NAP) is described. NAP improves accuracy of in silico fragmentation analyses by reranking candidate structures based on the network topology from MS/MS-based molecular networking. Annotation for the MS/MS spectral network of the Sageratia theezans twig extract was performed using NAP, and most molecular families within the network, including the known triterpenoids 1-7, could be putatively annotated, without relying on any previous reports of molecules from this species. Based on the in silico dereplication results, molecules were prioritized for isolation. In total, six dicoumaroyl 8- O-4' neolignans (8-13) and three dicoumaroyl lignans (14-16) were isolated from the twigs of S. theezans and structurally characterized by spectroscopic analyses. Isolates were evaluated for their neuroprotective activity, and compounds 14-16 showed potent protective effects against glutamate-induced oxidative stress in mouse HT22 cells at a concentration of 12.5 µM.

Propagating annotations of molecular networks using in silico fragmentation.

The annotation of small molecules is one of the most challenging and important steps in untargeted mass spectrometry analysis, as most of our biological interpretations rely on structural annotations. Molecular networking has emerged as a structured way to organize and mine data from untargeted tandem mass spectrometry (MS/MS) experiments and has been widely applied to propagate annotations. However, propagation is done through manual inspection of MS/MS spectra connected in the spectral networks and is only possible when a reference library spectrum is available. One of the alternative approaches used to annotate an unknown fragmentation mass spectrum is through the use of in silico predictions. One of the challenges of in silico annotation is the uncertainty around the correct structure among the predicted candidate lists. Here we show how molecular networking can be used to improve the accuracy of in silico predictions through propagation of structural annotations, even when there is no match to a MS/MS spectrum in spectral libraries. This is accomplished through creating a network consensus of re-ranked structural candidates using the molecular network topology and structural similarity to improve in silico annotations. The Network Annotation Propagation (NAP) tool is accessible through the GNPS web-platform https://gnps.ucsd.edu/ProteoSAFe/static/gnps-theoretical.jsp.

Three-Dimensional Microbiome and Metabolome Cartography of a Diseased Human Lung.

Our understanding of the spatial variation in the chemical and microbial makeup of an entire human organ remains limited, in part due to the size and heterogeneity of human organs and the complexity of the associated metabolome and microbiome. To address this challenge, we developed a workflow to enable the cartography of metabolomic and microbiome data onto a three-dimensional (3D) organ reconstruction built off radiological images. This enabled the direct visualization of the microbial and chemical makeup of a human lung from a cystic fibrosis patient. We detected host-derived molecules, microbial metabolites, medications, and region-specific metabolism of medications and placed it in the context of microbial distributions in the lung. Our tool further created browsable maps of a 3D microbiome/metabolome reconstruction map on a radiological image of a human lung and forms an interactive resource for the scientific community.

Coupling Targeted and Untargeted Mass Spectrometry for Metabolome-Microbiome-Wide Association Studies of Human Fecal Samples.

Increasing appreciation of the gut microbiome's role in health motivates understanding the molecular composition of human feces. To analyze such complex samples, we developed a platform coupling targeted and untargeted metabolomics. The approach is facilitated through split flow from one UPLC, joint timing triggered by contact closure relays, and a script to retrieve the data. It is designed to detect specific metabolites of interest with high sensitivity, allows for correction of targeted information, enables better quantitation thus providing an advanced analytical tool for exploratory studies. Procrustes analysis revealed that untargeted approach provides a better correlation to microbiome data, associating specific metabolites with microbes that produce or process them. With the subset of over one hundred human fecal samples from the American Gut project, the implementation of the described coupled workflow revealed that targeted analysis using combination of single transition per compound with retention time misidentifies 30% of the targeted data and could lead to incorrect interpretations. At the same time, the targeted analysis extends detection limits and dynamic range, depending on the compounds, by orders of magnitude. A software application has been developed as a part of the workflow to allows for quantitative assessments based on calibration curves. Using this approach, we detect expected microbially modified molecules such as secondary bile acids and unexpected microbial molecules including Pseudomonas-associated quinolones and rhamnolipids in feces, setting the stage for metabolome-microbiome-wide association studies (MMWAS).

Prioritizing Natural Product Diversity in a Collection of 146 Bacterial Strains Based on Growth and Extraction Protocols.

In order to expedite the rapid and efficient discovery and isolation of novel specialized metabolites, while minimizing the waste of resources on rediscovery of known compounds, it is crucial to develop efficient approaches for strain prioritization, rapid dereplication, and the assessment of favored cultivation and extraction conditions. Herein we interrogated bacterial strains by systematically evaluating cultivation and extraction parameters with LC-MS/MS analysis and subsequent dereplication through the Global Natural Product Social Molecular Networking (GNPS) platform. The developed method is fast, requiring minimal time and sample material, and is compatible with high-throughput extract analysis, thereby streamlining strain prioritization and evaluation of culturing parameters. With this approach, we analyzed 146 marine Salinispora and Streptomyces strains that were grown and extracted using multiple different protocols. In total, 603 samples were analyzed, generating approximately 1.8 million mass spectra. We constructed a comprehensive molecular network and identified 15 molecular families of diverse natural products and their analogues. The size and breadth of this network shows statistically supported trends in molecular diversity when comparing growth and extraction conditions. The network provides an extensive survey of the biosynthetic capacity of the strain collection and a method to compare strains based on the variety and novelty of their metabolites. This approach allows us to quickly identify patterns in metabolite production that can be linked to taxonomy, culture conditions, and extraction methods, as well as informing the most valuable growth and extraction conditions.